The present invention generally relates to new pharmacological formulations of phospholipid microbubbles comprising a plurality of phospholipids that allow for the rapid, efficient and target delivery of polyunsaturated fatty acids (“PUFA”) to areas of disease or injury in a subject for diagnostic, prophylactic and/or therapeutic aims.
Omega-3 PUFA:
Omega-3 PUFA is an essential nutrient critical for the maintenance of human health. Interest in omega-3 PUFA stems from the first observations by Bang and Dyerberg during the 1970s that death from coronary artery disease (CAD) is extremely rare among the Greenland Eskimos who consume a diet rich in EPA and DHA. Since that seminal discovery, intense basic and animal research has revealed a multitude of beneficial effects of omega-3 PUFA. These positive effects include anti-inflammatory, anti-cancerous, immunomodulatory, anti-diabetic, anti-thrombotic and anti-arrhythmic properties, to name but a few. In addition, numerous epidemiological studies and randomized controlled trials have been conducted that demonstrated similar wide-ranging health benefits in humans. For example, the landmark GISSI-P (Gruppo Italiano per lo Studio della Streptochinasi nell'Infarto Miocardico-Prevenzione) prevention study randomized 11,324 patients with pre-existing CAD to 850 mg of omega-3 PUFA resulted in a 45% reduction in sudden cardiac death and 30% reduction in cardiovascular death. (Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. The Lancet, 1999; 354:447-55). Moreover, the benefits of these essential nutrients appear not to be limited solely to the cardiovascular system but extend to virtually every organ system in the human body. (Simopoulos A P. Omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr 2002; 21:495-505).
Tragically, the dietary practices of modern industrial society over the last century have been moving towards a diet that is severely deficient in omega-3 PUFA. This nutritional deficit is further compounded by the rise in the consumption of “processed” foods containing hydrogenated trans-fatty acids and omega-6 PUFA that possess antagonistic properties to omega-3 PUFA. Moreover, the content of omega-3 PUFA in meat products was decreased by the modern practice of raising animals fed with corn-base stock in feedlots rather than grass fed on the open field. Together, these changes resulted in a striking imbalance of pro-inflammatory and anti-inflammatory types of PUFA in the typical modern diet. Up until only recently, humans evolved and flourished on a diet with a ratio of omega-6:omega-3 fatty acids close to 1:1. It has been estimated that today this ratio has risen dramatically to as high as 20:1. (Simopoulos A P. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomed Pharmacother 2002; 56:365-79). As a consequence of these changes in eating habits, food processing and in agriculture, entire populations are now at risk of developing a wide spectrum of inflammatory, autoimmune and degenerative diseases.
Despite their wide ranging health benefits, the therapeutic use of omega-3 PUFA is currently limited by the lack of a rapid and effective means of delivery. Oral supplementation of omega-3 PUFA requires weeks to months to attain adequate blood levels and to be incorporated into cellular membranes to exert their effects. Furthermore, the “fishy” unpleasant taste of omega-3 PUFA makes it difficult for patients to maintain strict compliance. More importantly, the delayed onset of action of oral supplementation has precluded their use in the treatment of acute emergent medical conditions such as acute myocardial infarction and cerebral vascular accidents and their complications. To achieve a more rapid onset of action, intravenous infusion of omega-3 PUFA has been used successfully to terminate and prevent life-threatening ventricular arrhythmias that occurred in the setting of AMI in various animal models and in humans. (McGuinness J, Neilan T G, Sharkasi A, Bouchier-Hayes D, Redmond J M. Myocardial protection using an omega-3 fatty acid infusion: Quantification and mechanism of action. J Thorn Cardiovasc Surg 2006; 132:72-9; Billman G E, Hallaq H, Leaf A. Prevention of Ischemia-Induced Ventricular Fibrillation by {omega}3 Fatty Acids. PNAS 1994; 91:4427-30; and Schrepf R, Limmert T, Claus Weber P, Theisen K, Sellmayer A. Immediate effects of n-3 fatty acid infusion on the induction of sustained ventricular tachycardia. Lancet 2004; 363:1441-2). Omega-3 PUFA have also been shown to be exert immuno-modulatory activity in preventing heart transplant rejection in rats. (Grimminger F, Grimm H, Fuhrer D, et al. {omega}-3 Lipid Infusion in a Heart Allotransplant Model: Shift in Fatty Acid and Lipid Mediator Profiles and Prolongation of Transplant Survival. Circulation 1996; 93:365-71). However, omega-3 PUFA administered by the intravenous route have to be in the form of triglycerides rather than in their free bioactive non-esterified forms. Enzymatic hydrolysis of these triglycerides by endogenous lipases is needed for their release and activation. Because of interindividual differences in the rate of their metabolism, the blood levels of omega-3 PUFA after intravenous infusion were found to be highly variable. (Schrepf R, Limmert T, Claus Weber P, Theisen K, Sellmayer A. Immediate effects of n-3 fatty acid infusion on the induction of sustained ventricular tachycardia. Lancet 2004; 363:1441-2). Because of the slow and variable rate of hydrolysis and the need to saturate the entire body, a prolonged infusion that lasts an excess of 90 minutes is generally required to ensure that adequate therapeutic levels can be achieved. This unpredictable bioavailability prevents their use in emergent clinical situations such as heart attacks, strokes and their arrhythmic sequela.
Ultrasound Microbubbles:
Prior art ultrasound microbubbles are gas-filled vesicles having diameters on the order of less than 10 microns enclosed in a biocompatible shell composed of a lipid, protein or polymer. The gas core is made of an inert high-molecular weight gas (i.e, perfluorocarbons, sulfur hexafluoride) such as to typically minimize volume loss and to ensure stability. The small size of the microbubbles allow for their unimpeded passage through the microcirculation of the lungs to any organs of the body by intravenous administration. Various formulations of these microbubbles are currently employed as contrast agents to enhance diagnostic images obtained by ultrasonography, as in echocardiography to help visualize the left ventricular cavity of the heart and to assess myocardial perfusion.
More recently, these microbubbles have been modified for therapeutic use as vehicles for drug delivery and for gene therapy. (Feinstein S B. The powerful microbubble: from bench to bedside, from intravascular indicator to therapeutic delivery system, and beyond. Am J Physiol Heart Circ Physiol 2004; 287:H450-7). The dual versatility of microbubbles for molecular imaging and target drug delivery termed “theranostic” applications is only now beginning to be exploited. (Pan D, Lanza G M, Wickline S A, Caruthers S D. Nanomedicine: Perspective and promises with ligand-directed molecular imaging. European Journal of Radiology 2009; 70:274-85). By focusing the ultrasound energy at a desired target site, higher local concentrations of a therapeutic agent may be achieved. For example, U.S. Pat. No. 5,558,092 describes compositions, methods and apparatus for carrying out diagnostic and therapeutic ultrasound. Contrast materials loaded with a therapeutic agent are imaged using diagnostic ultrasound waves, and once seen accumulating in a desired area, are ruptured using ultrasonic waves to generate enhanced cavitation or the targeted release of an agent into the region. Coupling diagnostic and therapeutic ultrasound modes provides additional advantages of monitoring efficacy and dose adjustments. However, the major limiting aspect of the current art remains poor efficiency of delivery.